CN110596407A - Preparation method for constructing electrochemical immunosensor based on mussel-like chemistry - Google Patents

Preparation method for constructing electrochemical immunosensor based on mussel-like chemistry Download PDF

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CN110596407A
CN110596407A CN201911063449.1A CN201911063449A CN110596407A CN 110596407 A CN110596407 A CN 110596407A CN 201911063449 A CN201911063449 A CN 201911063449A CN 110596407 A CN110596407 A CN 110596407A
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陈厚
王娜
柏良久
王文香
赵晓青
范德超
杨华伟
魏东磊
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Ludong University
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Abstract

The invention discloses a method for constructing an electrochemical immunosensor by utilizing a multi-wall carbon nano tube mussel-like chemical modification synthesis functional nano composite probe, which comprises the steps of firstly coating a polydopamine layer on the surface of a multi-wall carbon nano tube by utilizing mussel-like excitation chemistry, then grafting a bromine initiator, initiating by utilizing light-induced electron transfer-atom transfer free radical polymerization to form poly glycidyl methacrylate with controllable molecular weight on the surface of the multi-wall carbon nano tube, synthesizing a functional nano composite material by amino modification, and loading a large amount of electrochemically labeled anthraquinone dicarboxylic acid and ferrocene formate on a functional group of the functional nano composite material to prepare two nano composite probes for signal amplification. And finally, preparing an electrochemical immunosensor by means of the two functionalized nano composite probes, and performing electrochemical analysis and combined detection on carcinoembryonic antigens and alpha fetoprotein serving as two tumor markers.

Description

Preparation method for constructing electrochemical immunosensor based on mussel-like chemistry
Technical Field
The invention relates to a preparation method of an electrochemical immunosensor, in particular to a preparation method of a multi-walled carbon nanotube nano-composite probe designed based on simulated mussel chemistry and surface-initiated atom transfer radical polymerization technology and used for constructing the electrochemical immunosensor, and belongs to the technical field of functional polymers.
Background
The detection of Tumor Markers (TMs) is an important method and means for clinical diagnosis of cancer. However, since the serum content of TMs in early cancer patients is extremely low, there is an urgent need to expand the detection signal and improve the sensitivity of detection. With the progress of research, researchers find that one tumor may secrete multiple tumor markers, so that a single detection cannot meet the current situation. Therefore, some tumor markers which have higher specificity and can be complemented are often selected for combined determination, and the detection rate of the tumor is improved. Nowadays, expanding detection signals and joint detection have become the research hotspots for detecting tumor markers.
The common methods for detecting tumor markers include enzyme-linked immunosorbent assay, surface plasma resonance imaging, and chemiluminescence immunoassay. Compared with other detection methods, the electrochemical immunosensor has attracted extensive attention due to the advantages of high sensitivity, simple operation, low cost and the like, and has become one of important means for detecting TMs.
At present, the application of the nano material in the construction of the electrochemical immunosensor is reported. The introduction of the nano material in the electrochemical analysis mainly improves the conductivity and the specific surface area of the electrode and improves the catalytic activity of the electrode, thereby improving the detection sensitivity of the immunosensor and the real-time monitoring of the detected object. The multi-walled carbon nanotubes (MWCNTs) have good conductivity, and meanwhile, the large specific surface area of the MWCNTs is beneficial to immobilization of antibodies and promotion of electron transfer between active centers of the antibodies and the surfaces of electrodes. These advantages determine its good application prospects in the field of electrochemical analysis. However, it has been found that the use of nanomaterial alone as a carrier may not provide sufficient effective sites for loading signal molecules, and the expected purpose of amplifying the detection signal is not achieved. The problem can be effectively solved by modifying the nano material by utilizing surface modification and synthesizing functional high molecular polymer. The mussel-like polydopamine chemical attracts much attention, can be coated on any object through oxidative autopolymerization of dopamine, has a large number of amino and hydroxyl functional groups, and can effectively improve the surface performance of the multi-walled carbon nanotube. At present, polymerization methods of functional polymer brushes have been reported, wherein surface-initiated photoinduced electron transfer-atom transfer radical polymerization (PET-ATRP) has received much attention due to its concept of responding to green chemistry without requiring a large amount of copper catalyst and without contaminating the final polymer. Therefore, the MWCNTs are subjected to biomimetic mussel polydopamine modification and then grafted with the functional polymer brush to synthesize the nano probe, so that the electrochemical immunosensor with more excellent construction performance is constructed.
The invention relates to a bionic mussel polydopamine chemical-dopamine oxidizable and auto-polymerizable coating method, which comprises the steps of coating a polydopamine layer (PDA) on the surface of MWCNTs, connecting alpha-bromobenzene acetic acid on the surface of the MWCNTs through esterification reaction to form a macroinitiator, initiating molecular weight-controllable Poly Glycidyl Methacrylate (PGMA) through PET-ATRP under the illumination condition, introducing signal molecules anthraquinone-2-carboxylic acid (Aq) and ferrocene formate (Fc) after ethylenediamine modification, synthesizing a functionalized nano composite probe MWCNTs @ PDA-PGMA-Aq/Fc for signal amplification, and finally using the functionalized nano composite probe MWCNTs @ PDA-PGMA-Aq/Fc for constructing an electrochemical immunosensor. The functional nano composite probe prepared by the method can realize the combined high-sensitivity detection of tumor markers carcinoembryonic antigen (CEA) and Alpha Fetoprotein (AFP), and can undoubtedly greatly improve the early diagnosis rate of tumors.
Disclosure of Invention
The invention aims to provide a method for designing a multi-wall carbon nano tube nano composite probe by using simulated mussel chemistry and a surface initiated atom transfer radical polymerization technology and constructing an electrochemical immunosensor by using the multi-wall carbon nano tube nano composite probe.
In order to achieve the purpose, the specific technical scheme of the invention is as follows:
1. a preparation method for constructing an electrochemical immunosensor based on simulated mussel chemistry is characterized by comprising the following steps:
(1) the nano composite probe is designed by utilizing the mussel-like chemistry and the surface-initiated atom transfer radical polymerization technology:
firstly, a multi-wall carbon nano tube is chemically modified by using a mussel-like material: dissolving multi-walled carbon nanotubes (MWCNTs) in a mixed solution of ethanol and distilled water, performing ultrasonic dispersion, then adding dopamine hydrochloride (DA) under magnetic stirring, stirring for a period of time, dropwise adding a Tris buffer solution into a reaction system for reaction, finally washing with ethanol and distilled water, performing centrifugal separation to obtain a product MWCNTs @ PDA,
wherein the MWCNTs, DA, ethanol, water, Tris = 0.1 ~ 1:0.1 ~ 0.8.8: 1 ~ 2:1 ~ 1.5.5: 1 ~ 3, the reaction temperature is 25-30 ℃, the reaction time is 24 hours,
the pH value of the Tris buffer solution is 8.5, and the stirring speed is 300 ~ 500 rpm;
dispersing the obtained MWCNTs @ PDA in anhydrous Dichloromethane (DCM) for ultrasonic treatment, adding 4-Dimethylaminopyridine (DMAP) and bromophenylacetic acid (BPA), dissolving Dicyclohexylcarbodiimide (DCC) in the DCM under the stirring of an ice bath, dropwise adding the obtained solution into a round-bottom flask by using a constant-pressure dropping funnel, gradually increasing the temperature of a reaction system to room temperature, reacting for a certain time, centrifugally washing to collect a product MWCNTs @ PDA-Br,
wherein, the MWCNTs @ PDA: DMAP: BPA: DCC: DCM = 1:0.1 ~ 0.2:3 ~ 5:3 ~ 5:1 ~ 20,
the reaction solvent is as follows: anhydrous dichloromethane, esterification catalyst: 4-dimethyl amino pyridine, and a preparation method thereof,
the water loss agent in the reaction is dicyclohexylcarbodiimide, the reaction temperature is 15 ~ 35 ℃,
the reaction time is as follows: 4-24 hours;
secondly, functional polymer brushes are grafted on the surfaces of MWCNTs @ PDA-Br by utilizing the surface-initiated PET-ATRP technology, and Glycidyl Methacrylate (GMA), MWCNTs @ PDA-Br, Eosin Y (EY), Triethylamine (TEA) and alpha-bromophenylacetic acid are prepared in a bottle with a branch mouthEthyl Ester (EBPA) andN,N-Dimethylformamide (DMF) by charging the reactants with N2To achieve oxygen free N2The mixture is transferred into a magnetic driving stirring device containing an LED flexible strip, the reaction is carried out at room temperature overnight, the mixture after the reaction is dissolved in DMF, the mixture is precipitated into a large amount of methanol solution, the mixture is dialyzed after being centrifugally collected and then is frozen and dried to obtain the product MWCNTs @ PDA-PGMA,
wherein, the macroinitiator comprises glycidyl methacrylate, eosin Y, triethylamine, alpha-ethyl bromophenylacetate:N,N-dimethylformamide = 0.1 ~ 0.5.5: 1 ~ 10:0.1:1 ~ 3:0.1 ~ 1:1 ~ 20,
the PET-ATRP initiator comprises: MWCNTs @ PDA-Br, wherein the photocatalyst is: as a result of the eosin Y being present,
monomer (b): glycidyl methacrylate, sacrificial initiator: alpha-ethyl bromobenzene acetate, and the preparation method thereof,
reducing agent: triethylamine, solvent: n, N-dimethylformamide;
finally, preparing a functional nano composite probe by using the obtained MWCNTs @ PDA-PGMA, modifying the MWCNTs @ PDA-PGMA functional polymer brush by using Ethylenediamine (EDA), placing the functional PGMA polymer brush in a flask containing the EDA, stirring for a period of time, centrifuging and washing to obtain a final product,
wherein the reaction temperature is 50 ~ 55 ℃,
the reaction time is as follows: the reaction time is 24 hours,
then grafting two signal molecules, namely anthraquinone-2-carboxylic acid and ferrocene formate, on the obtained product, firstly placing the signal molecules in a phosphate buffer solution for full dispersion, adding a Dicyclohexylcarbodiimide (DCC)/4-Dimethylaminopyridine (DMAP) activating reagent to activate the carboxyl of the signal molecules, reacting at room temperature, then mixing with an ethylenediamine modified polymer brush, reacting at room temperature for 24 hours, centrifuging and freeze-drying to prepare the nano probe,
wherein the reaction temperature is: 20-30 ℃, catalytic agent of carboxyl: 4-Dimethylaminopyridine (DMAP), dehydration condensation agent: dicyclohexylcarbodiimide (DCC), solvent: phosphate buffer solution.
(2) Preparation of electrochemical immunosensor based on nano composite probe designed in (1)
Reacting the nano composite probe (MWCNTs @ PDA-PGMA-Aq/Fc) obtained in the step (1) with an antibody 2 of carcinoembryonic antigen (CEA) and Alpha Fetoprotein (AFP) in a shaking box at a certain temperature for 2 hours, polishing a glassy carbon electrode, assembling the antibody 1 (CEA + AFP), Bovine Serum Albumin (BSA), antigen (CEA + AFP) and the composite probe, performing electrochemical test,
wherein the nanocomposite probe: MWCNTs @ PDA-PGMA-Aq/Fc, the reaction temperature: 37 degrees celsius, CEA antibody 1 concentration: 0.01 mg/ml, AFP antibody 1 concentration: 0.01 mg/ml of the solution,
CEA antibody 2 concentration: 0.01 mg/ml, AFP antibody 2 concentration: 0.01 mg/ml of the solution,
BSA concentration of zero ~ percent,
163 femtograms/ml ~ 163 ng/ml of CEA antigen concentration, 100 femtograms/ml ~ 100 ng/ml of AFP antigen concentration,
the functionalized nano composite probe prepared by the method is applied to an immunosensor to realize the ultra-sensitive detection of tumor markers CEA and AFP, and the exponential amplification of signals is realized for the detection of the tumor markers.
The electrochemical test is as follows: the method comprises the steps of optimizing a cyclic voltammetry curve and an impedance curve in the assembling process, optimizing electrochemical testing conditions (pH of electrolyte, antigen incubation time and concentration of bovine serum albumin), combining a high-sensitivity detection curve and a linear relation curve, and testing anti-interference and stability.
The reaction system provides a method for assembling and constructing an electrochemical immunosensor by utilizing a nano composite probe prepared by using simulated mussel excitation chemistry, so the invention claims to protect the method for assembling and constructing the electrochemical immunosensor by utilizing the nano composite probe prepared by using the simulated mussel excitation chemistry, and the method mainly comprises the following steps:
(1) the preparation of the nanocomposite probe according to claim 1 and the detection method using the same,
(2) the nano composite probe is applied to the construction of an electrochemical immunosensor and the combined high-sensitivity electrochemical detection of a tumor marker, so that the expansion of a detection range and the reduction of a detection limit are realized.
Detailed Description
The invention is further described below with reference to exemplary embodiments, but the invention is not limited to the following embodiments. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.
The first embodiment is as follows: preparation of mussel-like chemically modified multi-walled carbon nanotube (MWCNTs @ PDA)
MWCNTs (100 mg) were dissolved in a mixed solution of ethanol (40 ml) and distilled water (30 ml), ultrasonically dispersed for 30 minutes, and then dopamine hydrochloride (80 mg) was added under magnetic stirring. After 10 minutes, Tris buffer (pH = 8.5,50 ml) was added dropwise to the reaction system, and reacted at room temperature for 24 hours. Finally, washing with ethanol and distilled water and centrifuging to obtain the product.
Example two: preparation of macroinitiator (MWCNTs @ PDA-Br)
The dried MWCNTs @ PDA (100 mg) was dissolved in anhydrous Dichloromethane (DCM) (20 ml) and sonicated for 30 min. Dimethylaminopyridine (DMAP) (20 mg) and bromobenzoic acid (BPA) (340 mg) were then added. Dicyclohexylcarbodiimide (DCC) (330 mg) was dissolved in DCM (10 ml) with stirring in an ice bath, added dropwise to the round-bottom flask using an isopiestic dropping funnel, and the temperature of the reaction was gradually raised to room temperature. After 24 hours, it was collected by centrifugation and washed several times with ethanol and distilled water, and finally the product was dried.
Example three: preparation of surface-initiated PET-ATRP functional Polymer Brush (MWCNTs @ PDA-PGMA)
Prepared from GMA (6.04 mL, 45.6 mmol), MWCNTs @ PDA-Br (50 mg), EY (15.8 mg, 0.0228 mmol), TEA (95. mu.L, 0.684 mmol), EBPA (40. mu.L, 0.228 mmol) and DMF (20 mmol) in a split vialLiter) of the reaction mixture. By charging the reactants with N2To achieve oxygen free N2An atmosphere. The mixture was then transferred to a magnetically driven stirring apparatus containing LED flexible strips for LED illumination (lambda)max= 460 nm), the reaction was performed overnight at room temperature. The reaction was exposed to air and terminated in the dark. The reaction mixture was then dissolved in DMF and precipitated into a large volume of methanol solution. Centrifuging to collect the lower layer precipitate, washing with DMF and water, dialyzing, and freeze drying to obtain the product.
Example four: preparation of functionalized nano composite probe (MWCNTs @ PDA-PGMA-Aq/Fc)
MWCNTs @ PDA @ PGMA (10 mg) was placed in a flask containing EDA (5 mL), followed by stirring at 50 ℃ for 24 hours, and the final product, MWCNTs @ PDA-PGMA-EDA, was centrifuged, washed with ethanol and distilled water, and freeze-dried.
The polymer brush is grafted with two signal molecules after being modified by ethylenediamine. Firstly, the signal molecules are placed in phosphate buffer solution for full dispersion, DCC/DMAP activating reagent is added, and the reaction is carried out for 2 hours at room temperature. Then mixed with the ethylenediamine modified polymer, reacted for 24 hours at room temperature, centrifuged and lyophilized for further use. Reacting the MWCNTs @ PDA-PGMA-Aq + Fc grafted with the signal molecules with an antibody 2 (anti-CEA + anti-AFP) in a shaking box at 37 ℃ for 2 hours. And then centrifuging the mixture for 40 minutes by using a 4-DEG C refrigerated centrifuge, putting the lower-layer solid into a phosphate buffer solution, and putting the solution into a refrigerator for storage. And finally, polishing the glassy carbon electrode, and assembling an electrochemical immunosensor by using an antibody 1 (anti-CEA + anti-AFP), Bovine Serum Albumin (BSA), an antigen (CEA + AFP) and a composite probe layer by layer to be applied to combined super-sensitivity detection of the CEA and AFP.

Claims (2)

1. A preparation method for constructing an electrochemical immunosensor based on simulated mussel chemistry is characterized by comprising the following steps:
(1) the nano composite probe is designed by utilizing the mussel-like chemistry and the surface-initiated atom transfer radical polymerization technology:
firstly, a multi-wall carbon nano tube is chemically modified by using a mussel-like material: dissolving multi-walled carbon nanotubes (MWCNTs) in a mixed solution of ethanol and distilled water, performing ultrasonic dispersion, then adding dopamine hydrochloride (DA) under magnetic stirring, stirring for a period of time, dropwise adding a Tris buffer solution into a reaction system for reaction, finally washing with ethanol and distilled water, performing centrifugal separation to obtain a product MWCNTs @ PDA,
dispersing the obtained MWCNTs @ PDA in anhydrous Dichloromethane (DCM) for ultrasonic treatment, adding 4-Dimethylaminopyridine (DMAP) and bromophenylacetic acid (BPA), dissolving Dicyclohexylcarbodiimide (DCC) in the DCM under the stirring of an ice bath, dropwise adding the obtained solution into a round-bottom flask by using a constant-pressure dropping funnel, gradually increasing the temperature of a reaction system to room temperature, reacting for a certain time, centrifugally washing to collect a product MWCNTs @ PDA-Br,
the reaction solvent is as follows: anhydrous dichloromethane, esterification catalyst: 4-dimethyl amino pyridine, and a preparation method thereof,
the water loss agent in the reaction is dicyclohexylcarbodiimide, the reaction temperature is 15 ~ 35 ℃,
the reaction time is as follows: 4-24 hours;
secondly, functional polymer brushes are grafted on the surfaces of MWCNTs @ PDA-Br by utilizing the surface-initiated PET-ATRP technology, and Glycidyl Methacrylate (GMA), MWCNTs @ PDA-Br, Eosin Y (EY), Triethylamine (TEA), alpha-bromobenzene ethyl acetate (EBPA) andN,N-Dimethylformamide (DMF) by charging the reactants with N2To achieve oxygen free N2The mixture is transferred into a magnetic driving stirring device containing an LED flexible strip, the reaction is carried out at room temperature overnight, the mixture after the reaction is dissolved in DMF, the mixture is precipitated into a large amount of methanol solution, the mixture is dialyzed after being centrifugally collected and then is frozen and dried to obtain the product MWCNTs @ PDA-PGMA,
the PET-ATRP initiator comprises: MWCNTs @ PDA-Br, wherein the photocatalyst is: as a result of the eosin Y being present,
monomer (b): glycidyl methacrylate, sacrificial initiator: alpha-ethyl bromobenzene acetate, and the preparation method thereof,
reducing agent: triethylamine, solvent: n, N-dimethylformamide;
finally, preparing a functional nano composite probe by using the obtained MWCNTs @ PDA-PGMA, modifying the MWCNTs @ PDA-PGMA functional polymer brush by using Ethylenediamine (EDA), placing the functional PGMA polymer brush in a flask containing the EDA, stirring for a period of time, centrifuging and washing to obtain a final product,
wherein the reaction temperature is 50 ~ 55 ℃,
the reaction time is as follows: the reaction time is 24 hours,
then grafting two signal molecules, namely anthraquinone-2-carboxylic acid and ferrocene formate, on the obtained product, firstly placing the signal molecules in a phosphate buffer solution for full dispersion, adding a Dicyclohexylcarbodiimide (DCC)/4-Dimethylaminopyridine (DMAP) activating reagent to activate the carboxyl of the signal molecules, reacting at room temperature, then mixing with an ethylenediamine modified polymer brush, reacting at room temperature for 24 hours, centrifuging and freeze-drying to prepare the nano probe,
wherein the reaction temperature is: 20-30 ℃, catalytic agent of carboxyl: 4-Dimethylaminopyridine (DMAP), dehydration condensation agent: dicyclohexylcarbodiimide (DCC), solvent: a phosphate buffer solution;
(2) reacting the nano composite probe (MWCNTs @ PDA-PGMA-Aq/Fc) obtained in the step (1) with an antibody 2 of carcinoembryonic antigen (CEA) and Alpha Fetoprotein (AFP) in a shaking box at a certain temperature for 2 hours, polishing a glassy carbon electrode, assembling the antibody 1 (CEA + AFP), Bovine Serum Albumin (BSA), antigen (CEA + AFP) and the composite probe, performing electrochemical test, preparing the functionalized nano composite probe by the method, applying the functionalized nano composite probe to an immunosensor to realize the ultrasensitive detection of tumor markers CEA and AFP, realizing the exponential amplification of signals for the detection of the tumor markers,
wherein the BSA concentration is two percent of zero ~ percent, the CEA antigen concentration is 163 femtograms/ml ~ 163 ng/ml, and the AFP antigen concentration is 100 femtograms/ml ~ 100 ng/ml.
2. The polymer brush-based nano composite probe is prepared by applying the preparation process.
CN201911063449.1A 2019-11-04 2019-11-04 Preparation method for constructing electrochemical immunosensor based on mussel-like chemistry Pending CN110596407A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114354583A (en) * 2022-01-07 2022-04-15 河南中医药大学 Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof
CN114870798A (en) * 2022-03-04 2022-08-09 华北电力大学 Novel three-dimensional layered hierarchical mesoporous carbon material and preparation method thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114354583A (en) * 2022-01-07 2022-04-15 河南中医药大学 Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof
CN114354583B (en) * 2022-01-07 2024-06-07 河南中医药大学 Electrochemiluminescence lung cancer detection kit based on metal-free light ATRP signal amplification strategy, and use method and application thereof
CN114870798A (en) * 2022-03-04 2022-08-09 华北电力大学 Novel three-dimensional layered hierarchical mesoporous carbon material and preparation method thereof

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